DNA Scission Chemistry and EPR Studies of Four New Bis(2,6-Dimethoxyhydroquinone–3-Mercaptoacetic Acid)–Peptide Conjugates

Abstract

In an effort to investigate the use of short peptide chains as carriers of new antitumour agents, four tripeptide cytotoxic agent conjugates, namely DMQ–MA–Lys(DMQ–MA)–Lys(Cbz)–Arg–OMe, DMQ–MA–Lys(DMQ–MA)–Phe–Arg–OMe, DMQ–MA–Lys(DMQ–MA)–Ile–Arg–OMe, and DMQ–MA–Lys(DMQ–MA)–Val–Arg–OMe, were synthesized. The cytotoxic agent conjugated to the N-terminal, and the ξ-amino group of lysine of the tripeptides was 2,6-dimethoxyhydroquinone–3-mercaptoacetic acid (DMQ–MA). The tripeptides were synthesized by coupling protected amino acid residues in solution according to Pfp/DCC methods (where Pfp refers to pentafluorophenol and DCC refers to N,N^′-dicyclohexylcarbodiimide). DNA scission chemistry showed that these compounds were able to cleave supercoiled DNA into open-circular form in drug concentrations as low as 5–20 μM without addition of H₂O₂ or UV irradiation. DNA cleavage was clearly inhibited when hydroxyl radical scavengers such as glycerol and sodium benzoate were added to the reaction system. When the drugs were added to rhodamine B, the UV absorbance and fluorescence intensity of rhodamine B decreased quickly due to the degradation caused by the hydroxyl radicals. Electron paramagnetic resonance (EPR) experiments employing the spin-trap agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) showed that the signal intensity of the hydroxyl radicals generated from these newly synthesized DMQ–MA–peptide compounds was a non-linear function of ascorbic acid concentration. Deoxyribonucleic acid binding constants of these compounds were in the 10⁴ M^–1 range. It was concluded that DNA cleavage was not related to the ability of these drugs to produce hydroxyl radicals exclusively; indeed, it was related to their ability to produce various radicals and reactive species.